This project is focused on the cellular and molecular processes that occur during the differentiation of IgA B cells, the main type of B cell developing in mucosal lymphoid follicles. During this phase of our studies we continued our examination of the role of germline transcripts in isotype differentiation generally and IgA switching specifically. In particular, we gathered several pieces of evidence that isotype switching is associated with "transplicing", i.e., splicing of RNA transcribed from widely separated segments of the Ig gene. First, we demonstrated that sIgA+ B cell lymphoma cells (CH12.LX.A2 cells) which lack Ialpha gene segments and are transfected with plasmid constructs that can be induced to express Ialpha, produce Ialpha-Calpha (germline) transcripts. Since it is unlikely that the transfected Ialpha gene integrated at a site close to Calpha in all cells, this result strongly suggests that Ialpha is transpliced to Calpha in the transfected A2 B cells. Next, we showed that CH12.LX B cells, cells that constitutively produce Ialpha germline transcripts and spontaneously switch from sIgM+ to sIgA+ B cells, produce JH-Ialpha transpliced mRNA transcripts. The latter were shown with highly controlled RT-PCR techniques, followed by sequencing of the amplified cDNA. Finally, we showed that dexamethasone, an agent that induces sIgM+ CH12.LX B cells to differentiate into sIgM+/sIgA+ switch intermediate cells, also induces increased levels of JH2-Ialpha transpliced mRNA. Taken together, these data strongly suggest that CH12.LX B cells undergoing isotype switching pass through a stage during which they transplice a VDJ-Ialpha-Calpha message that is further processed to a VDJ-Calpha message prior to switch rearrangement. Such transpliced messages may "direct" subsequent DNA rearrangement.